1. Field of the Invention
This invention relates generally to antimicrobial agents, and more specifically pertains to peptides useful as antimicrobial agents for the prevention and treatment of infections caused by organisms, such as bacteria and fungi, many of which are resistant to conventional antibiotics.
2. Description of Related Art
Human infections due to antibiotic-resistant bacteria and fungi are increasing in frequency and severity. Microbial pathogens exhibiting resistance to one or more antibiotics can now commonly be found in community and nosocomial settings. Antibiotic resistant pathogens currently of the greatest concern are methicillin (multiple) resistant Staphylococcus aureus (MRSA), vancomycin intermediate-resistant S. aureus (VISA) or vancomycin-resistant S. aureus (VRSA), vancomycin resistant Enterococcus faecalis or Enterococcus faecium (VRE), multidrug-resistant Streptococcus pneumoniae (MDRSPn) or Streptococcus pyogenes (MDRSPy), multidrug-resistant Pseudomonas aeruginosa (MDRPA), and azole resistant Candida albicans (ARCA).
Antimicrobial peptides have heretofore generally been considered to have undesirable toxicity, immunogenicity, and short half-lives due to biodegradation. However, endogenous antimicrobial peptides are believed to be integral to non-oxidative mechanisms of antimicrobial host defense. Stable, peptide-resistant mutants are rare, likely because microbicidal peptides appear to target the cytoplasmic membrane or other essential structures and/or functions of pathogens.
Investigations conducted over the past decade have demonstrated the existence of potent microbicidal peptides from various mammalian tissues. Perhaps the most thoroughly studied among these are defensins from neutrophil azurophilic granules. Related peptides such as β-defensins and cryptdins have also been isolated and characterized. To date, nearly 20 distinct defensins have been found in mammalian neutrophils.
Aside from neutrophils, the probability that platelets play an integral role in host defense against infection has been demonstrated by the following facts: i) platelets are the earliest and predominant cells at sites of microbial infection of vascular endothelium; ii) platelets adhere to and internalize microbial pathogens; iii) bacterial, fungal, and protozoal pathogens are damaged or killed by activated platelets in vitro; iv) thrombocytopenia increases susceptibility to and severity of some infections; v) rabbit and human platelets release platelet microbicidal proteins (PMPs) when stimulated with microorganisms or platelet agonists integral to infection in vitro; and vi) PMPs exert rapid and potent microbicidal activities against a broad spectrum of pathogens in vitro. It has been hypothesized that PMPs substantially contribute to platelet antimicrobial host defense by direct microbicidal actions, and may amplify cell mediated immune mechanisms such as neutrophil microbicidal activity. Similar to defensins, PMPs appear to disrupt microbial cytoplasmic membranes to achieve microbicidal activity. Present data indicates that PMP-2 (Sequence No. 1), tPMP-1, and defensin hNP-1 employ distinct mechanisms, and that these differences are related to differences in protein structure.
The majority of known mammalian antimicrobial peptides have been localized within leukocytes (e.g., defensins), or secreted onto epithelial surfaces such as intestinal lumen or tracheal epithelium (e.g., cryptdins, tracheal antimicrobial peptide). Prohibitive levels of mammalian cell toxicity have been noted with many of these peptides when they have been tested as antimicrobial therapeutics. In contrast, PMPs exert potent in vitro microbicidal activity against a broad spectrum of bacteria and fungi under physiological conditions that exist in the intravascular space. Several PMPs are released from platelets stimulated with agonists associated with infection. Therefore, in response to tissue injury, PMPs are likely released into the mammalian bloodstream at localized sites of infection. In preliminary studies, tPMP-1 and PMP-2 (Sequence No. 1) have been found to cause minimal damage of human erythrocytes or vascular endothelial cells in vitro as compared with defensin hNP-1. In addition, PMPs exert potent microbicidal activity against bacterial and fungal pathogens, comparable to defensins which have been observed at concentrations as low as 0.5 μg/ml in vitro. These potencies compare favorably to potent conventional antimicrobial agents such as aminoglycosides or amphotericin B.
A large family of antimicrobial peptides from mammalian platelets has also been isolated, and amino acid compositions and primary structures of endogenous antimicrobial peptides originating from mammalian and non-mammalian tissues now constitute a database of over 300 antimicrobial peptides. Recent advances in peptide structural analyses have provided important new information regarding the relationship between structure and microbicidal activities among these peptides. For example, the fact that many antimicrobial peptides are small, cationic, and contain amphiphilic α-helical domains is well established.
It would be desirable to provide peptides that are active against organisms that exhibit resistance to antibiotics, for use either independently or in combination to potentiate conventional antimicrobial agents or other antimicrobial peptides and/or which potentiate the antimicrobial functions of leukocytes. It is also desirable to provide microbicidal peptides that are based upon natural antimicrobial peptides, to overcome problems of toxicity and immunogenicity. To overcome short half-life due to degradation, such peptides should be resistant to proteolytic degradation, and should be stable in temperatures as high as 80° C., and in extremes of alkalinity and acidity, ranging from about pH 2 to about pH 10, for example. It is further desirable that such peptides should be amenable to chemical synthesis or recombinant DNA-based expression, facilitating their production in quantities necessary for testing or therapeutic application. The present invention addresses, at least in part, these and other needs.